Biopsy of the sentinel lymph node provides a convenient prognostic indicator for breast cancer, melanoma, as well as other cancers. Localization of the sentinel or "closest" lymph node to the primary tumor involves nuclear imaging with technetium 99m sulfur colloid and enables minimal surgery and subsequent biopsy. In a sentinel lymph node biopsy procedure in the breast, technetium 99m sulfur colloid and isosulfan blue dye are injected peritumorally. The radiopharmaceutical and the blue dye then convect to the sentinel lymph nodes. Prior to surgery, the sentinel node is localized via a gamma camera and in surgery, is identified with a hand-held gamma probe. Also blue lymph nodes and/blue lymphatic channels, serve as visual, intraoperative guides. The presence of cancerous cells is subsequently determined by histopathology. Instead of using a radiopharmaceutical for contrast, we are developing near-infrared fluorescent contrast agents and an optical imaging procedure to perform sentinel lymph node mapping in vivo minimizing the radiation exposure to the patient as well as to clinicians. The ability to synthesize fluorescent agents that target cancer cells as well as "report" disease offer new molecular reporting methods which may reduce the need for surgical resection and subsequent biopsy. Yet with the exception of phantom studies, the ability to collect fluorescence emitted from the surface of clinically relevant tissue volumes has not been demonstrated.The milestones of this R21 portion for developing NIR fluorescence imaging are (1) to replicate a laboratory based imaging system for ambient light rejection; (2) to demonstrate comparable or superior performance to nuclear gamma camera imaging with metrics of target-to-background and signal to noise ratios; and (3) to evidence its dynamic use for tracking lymph flow through the sentinel node using a safe, FDA-approved nonspecific agent, indocyanine green (ICG), within a small group of breast cancer patients under going static imaging with technetium 99m sulfur colloid. The milestones of the R33 portion of the proposed technology are to further develop, implement, and evaluate the technology for developing an optical based method for sentinel lymph node assessment for (1) imaging within the nuclear suite as well as (2) within the operating theatre.